Genetic Inhibition of double-stranded RNA

ABSTRACT

A process is provided of introducing an RNA into a living cell to inhibit gene expression of a target gene in that cell. The process may be practiced ex vivo or in vivo. The RNA has a region with double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical. The present invention is distinguished from prior art interference in gene expression by antisense or triple-strand methods.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Appln. No.60/068,562, filed Dec. 23, 1997.

GOVERNMENT RIGHTS

This invention was made with U.S. government support under grant numbersGM-37706, GM-17164, HD-33769 and GM-07231 awarded by the NationalInstitutes of Health. The U.S. government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to gene-specific inhibition of geneexpression by double-stranded ribonucleic acid (dsRNA).

2. Description of the Related Art

Targeted inhibition of gene expression has been a long-felt need inbiotechnology and genetic engineering. Although a major investment ofeffort has been made to achieve this goal, a more comprehensive solutionto this problem was still needed.

Classical genetic techniques have been used to isolate mutant organismswith reduced expression of selected genes. Although valuable, suchtechniques require laborious mutagenesis and screening programs, arelimited to organisms in which genetic manipulation is well established(e.g., the existence of selectable markers, the ability to controlgenetic segregation and sexual reproduction), and are limited toapplications in which a large number of cells or organisms can besacrificed to isolate the desired mutation. Even under thesecircumstances, classical genetic techniques can fail to producemutations in specific target genes of interest, particularly whencomplex genetic pathways are involved. Many applications of moleculargenetics require the ability to go beyond classical genetic screeningtechniques and efficiently produce a directed change in gene expressionin a specified group of cells or organisms. Some such applications areknowledge-based projects in which it is of importance to understand whateffects the loss of a specific gene product (or products) will have onthe behavior of the cell or organism. Other applications are engineeringbased, for example: cases in which is important to produce a populationof cells or organisms in which a specific gene product (or products) hasbeen reduced or removed. A further class of applications istherapeutically based in which it would be valuable for a functioningorganism (e.g., a human) to reduce or remove the amount of a specifiedgene product (or products). Another class of applications provides adisease model in which a physiological function in a living organism isgenetically manipulated to reduce or remove a specific gene product (orproducts) without making a permanent change in the organism's genome.

In the last few years, advances in nucleic acid chemistry and genetransfer have inspired new approaches to engineer specific interferencewith gene expression. These approaches are described below.

Use of Antisense Nucleic Acids to Engineer Interference

Antisense technology has been the most commonly described approach inprotocols to achieve gene-specific interference. For antisensestrategies, stoichiometric amounts of single-stranded nucleic acidcomplementary to the messenger RNA for the gene of interest areintroduced into the cell. Some difficulties with antisense-basedapproaches relate to delivery, stability, and dose requirements. Ingeneral, cells do not have an uptake mechanism for single-strandednucleic acids, hence uptake of unmodified single-stranded material isextremely inefficient. While waiting for uptake into cells, thesingle-stranded material is subject to degradation. Because antisenseinterference requires that the interfering material accumulate at arelatively high concentration (at or above, the concentration ofendogenous mRNA), the amount required to be delivered is a majorconstraint on efficacy. As a consequence, much of the effort indeveloping antisense technology has been focused on the production ofmodified nucleic acids that are both stable to nuclease digestion andable to diffuse readily into cells. The use of antisense interferencefor gene therapy or other whole-organism applications has been limitedby the large amounts of oligonucleotide that need to be synthesized fromnon-natural analogs, the cost of such synthesis, and the difficulty evenwith high doses of maintaining a sufficiently concentrated and uniformpool of interfering material in each cell.

Triple-Helix Approaches to Engineer Interference

A second, proposed method for engineered interference is based on atriple helical nucleic acid structure. This approach relies on the rareability of certain nucleic acid populations to adopt a triple-strandedstructure. Under physiological conditions, nucleic acids are virtuallyall single- or double-stranded, and rarely if ever form triple-strandedstructures. It has been known for some time, however, that certainsimple purine- or pyrimidine-rich sequences could form a triple-strandedmolecule in vitro under extreme conditions of pH (i.e., in a test tube).Such structures are generally very transient under physiologicalconditions, so that simple delivery of unmodified nucleic acids designedto produce triple-strand structures does not yield interference. As withantisense, development of triple-strand technology for use in vivo hasfocused on the development of modified nucleic acids that would be morestable and more readily absorbed by cells in vivo. An additional goal indeveloping this technology has been to produce modified nucleic acidsfor which the formation of triple-stranded material proceeds effectivelyat physiological pH.

Co-Suppression Phenomena and Their Use in Genetic Engineering

A third approach to gene-specific interference is a set of operationalprocedures grouped under the name “co-suppression”. This approach wasfirst described in plants and refers to the ability of transgenes tocause silencing of an unlinked but homologous gene. More recently,phenomena similar to co-suppression have been reported in two animals:C. elegans and Drosophila. Co-suppression was first observed byaccident, with reports coming from groups using transgenes in attemptsto achieve over-expression of a potentially useful locus. In some casesthe over-expression was successful while, in many others, the result wasopposite from that expected. In those cases, the transgenic plantsactually showed less expression of the endogenous gene. Severalmechanisms have so far been proposed for transgene-mediatedco-suppression in plants; all of these mechanistic proposals remainhypothetical, and no definitive mechanistic description of the processhas been presented. The models that have been proposed to explainco-suppression can be placed in two different categories. In one set ofproposals, a direct physical interaction at the DNA- or chromatin-levelbetween two different chromosomal sites has been hypothesized to occur;an as-yet-unidentified mechanism would then lead to de novo methylationand subsequent suppression of gene expression. Alternatively, some havepostulated an RNA intermediate, synthesized at the transgene locus,which might then act to produce interference with the endogenous gene.The characteristics of the interfering RNA, as well as the nature of theinterference process, have not been determined. Recently, a set ofexperiments with RNA viruses have provided some support for thepossibility of RNA intermediates in the interference process. In theseexperiments, a replicating RNA virus is modified to include a segmentfrom a gene of interest. This modified virus is then tested for itsability to interfere with expression of the endogenous gene. Initialresults with this technique have been encouraging, however, theproperties of the viral RNA that are responsible for interferenceeffects have not been determined and, in any case, would be limited toplants which are hosts of the plant virus.

Distinction Between the Present Invention and Antisense Approaches

The present invention differs from antisense-mediated interference inboth approach and effectiveness. Antisense-mediated genetic interferencemethods have a major challenge: delivery to the cell interior ofspecific single-stranded nucleic acid molecules at a concentration thatis equal to or greater than the concentration of endogenous mRNA.Double-stranded RNA-mediated inhibition has advantages both in thestability of the material to be delivered and the concentration requiredfor effective inhibition. Below, we disclose that in the model organismC. elegans, the present invention is at least 100-fold more effectivethan an equivalent antisense approach (i.e., dsRNA is at least 100-foldmore effective than the injection of purified antisense RNA in reducinggene expression). These comparisons also demonstrate that inhibition bydouble-stranded RNA must occur by a mechanism distinct from antisenseinterference.

Distinction Between the Present Invention and Triple-Helix Approaches

The limited data on triple strand formation argues against theinvolvement of a stable triple-strand intermediate in the presentinvention. Triple-strand structures occur rarely, if at all, underphysiological conditions and are limited to very unusual base sequencewith long runs of purines and pyrimidines. By contrast, dsRNA-mediatedinhibition occurs efficiently under physiological conditions, and occurswith a wide variety of inhibitory and target nucleotide sequences. Thepresent invention has been used to inhibit expression of 18 differentgenes, providing phenocopies of null mutations in these genes of knownfunction. The extreme environmental and sequence constraints ontriple-helix formation make it unlikely that dsRNA-mediated inhibitionin C. elegans is mediated by a triple-strand structure.

Distinction Between Present Invention and Co-Suppression Approaches

The transgene-mediated genetic interference phenomenon calledco-suppression may include a wide variety of different processes. Fromthe viewpoint of application to other types of organisms, theco-suppression phenomenon in plants is difficult to extend. Aconfounding aspect in creating a general technique based onco-suppression is that some transgenes in plants lead to suppression ofthe endogenous locus and some do not. Results in C. elegans andDrosophila indicate that certain transgenes can cause interference(i.e., a quantitative decrease in the activity of the correspondingendogenous locus) but that most transgenes do not produce such aneffect. The lack of a predictable effect in plants, nematodes, andinsects greatly limits the usefulness of simply adding transgenes to thegenome to interfere with gene expression. Viral-mediated co-suppressionin plants appears to be quite effective, but has a number of drawbacks.First, it is not clear what aspects of the viral structure are criticalfor the observed interference. Extension to another system would requirediscovery of a virus in that system which would have these properties,and such a library of useful viral agents are not available for manyorganisms. Second, the use of a replicating virus within an organism toeffect genetic changes (e.g., long- or short-term gene therapy) requiresconsiderably more monitoring and oversight for deleterious effects thanthe use of a defined nucleic acid as in the present invention.

The present invention avoids the disadvantages of thepreviously-described methods for genetic interference. Severaladvantages of the present invention are discussed below, but numerousothers will be apparent to one of ordinary skill in the biotechnologyand genetic engineering arts.

SUMMARY OF THE INVENTION

A process is provided for inhibiting expression of a target gene in acell. The process comprises introduction of RNA with partial or fullydouble-stranded character into the cell or into the extracellularenvironment. Inhibition is specific in that a nucleotide sequence from aportion of the target gene is chosen to produce inhibitory RNA. Wedisclose that this process is (1) effective in producing inhibition ofgene expression, (2) specific to the targeted gene, and (3) general inallowing inhibition of many different types of target gene.

The target gene may be a gene derived from the cell, an endogenous gene,a transgene, or a gene of a pathogen which is present in the cell afterinfection thereof. Depending on the particular target gene and the doseof double stranded RNA material delivered, the procedure may providepartial or complete loss of function for the target gene. A reduction orloss of gene expression in at least 99% of targeted cells has beenshown. Lower doses of injected material and longer times afteradministration of dsRNA may result in inhibition in a smaller fractionof cells. Quantitation of gene expression in a cell may show similaramounts of inhibition at the level of accumulation of target mRNA ortranslation of target protein.

The RNA may comprise one or more strands of polymerized ribonucleotide;it may include modifications to either the phosphate-sugar backbone orthe nucleoside. The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. RNAduplex formation may be initiated either inside or outside the cell. TheRNA may be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses of double-stranded material may yield moreeffective inhibition. Inhibition is sequence-specific in that nucleotidesequences corresponding to the duplex region of the RNA are targeted forgenetic inhibition. RNA containing a nucleotide sequences identical to aportion of the target gene is preferred for inhibition. RNA sequenceswith insertions, deletions, and single point mutations relative to thetarget sequence have also been found to be effective for inhibition.Thus, sequence identity may optimized by alignment algorithms known inthe art and calculating the percent difference between the nucleotidesequences. Alternatively, the duplex region of the RNA may be definedfunctionally as a nucleotide sequence that is capable of hybridizingwith a portion of the target gene transcript.

The cell with the target gene may be derived from or contained in anyorganism (e.g., plant, animal, protozoan, virus, bacterium, or fungus).RNA may be synthesized either in vivo or in vitro. Endogenous RNApolymerase of the cell may mediate transcription in vivo, or cloned RNApolymerase can be used for transcription in vivo or in vitro. Fortranscription from a transgene in vivo or an expression construct, aregulatory region may be used to transcribe the RNA strand (or strands).

The RNA may be directly introduced into the cell (i.e.,intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing an organism in a solutioncontaining RNA. Methods for oral introduction include direct mixing ofRNA with food of the organism, as well as engineered approaches in whicha species that is used as food is engineered to express an RNA, then fedto the organism to be affected. Physical methods of introducing nucleicacids include injection directly into the cell or extra-cellularinjection into the organism of an RNA solution.

The advantages of the present invention include: the ease of introducingdouble-stranded RNA into cells, the low concentration of RNA which canbe used, the stability of double-stranded RNA, and the effectiveness ofthe inhibition. The ability to use a low concentration of anaturally-occurring nucleic acid avoids several disadvantages ofanti-sense interference. This invention is not limited to in vitro useor to specific sequence compositions, as are techniques based ontriple-strand formation. And unlike antisense interference,triple-strand interference, and co-suppression, this invention does notsuffer from being limited to a particular set of target genes, aparticular portion of the target gene's nucleotide sequence, or aparticular transgene or viral delivery method. These concerns have beena serious obstacle to designing general strategies according to theprior art for inhibiting gene expression of a target gene of interest.

Furthermore, genetic manipulation becomes possible in organisms that arenot classical genetic models. Breeding and screening programs may beaccelerated by the ability to rapidly assay the consequences of aspecific, targeted gene disruption. Gene disruptions may be used todiscover the function of the target gene, to produce disease models inwhich the target gene are involved in causing or preventing apathological condition, and to produce organisms with improved economicproperties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the genes used to study RNA-mediated genetic inhibition inC. elegans. Intron-exon structure for genes used to test RNA-mediatedinhibition are shown (exons: filled boxes; introns: open boxes; 5′ and3′ untranslated regions: shaded; unc-22⁹, unc-54¹², fem-1¹⁴, andhlh-1¹⁵).

FIGS. 2 A-I show analysis of inhibitory RNA effects in individual cells.These experiments were carried out in a reporter strain (called PD4251)expressing two different reporter proteins, nuclear GFP-LacZ andmitochondrial GFP. The micrographs show progeny of injected animalsvisualized by a fluorescence microscope. Panels A (young larva), B(adult), and C (adult body wall; high magnification) result frominjection of a control RNA (ds-unc22A). Panels D-F show progeny ofanimals injected with ds-gfpG. Panels G-I demonstrate specificity.Animals are injected with ds-lacZL RNA, which should affect the nuclearbut not the mitochondrial reporter construct. Panel H shows a typicaladult, with nuclear GFP-LacZ lacking in almost all body-wall muscles butretained in vulval muscles. Scale bars are 20 μm.

FIGS. 3 A-D show effects of double-stranded RNA corresponding to mex-3on levels of the endogenous mRNA. Micrographs show in situ hybridizationto embryos (dark stain). Panel A: Negative control showing lack ofstaining in the absence of hybridization probe. Panel B: Embryo fromuninjected parent (normal pattern of endogenous mex-3 RNA²⁰). Panel C:Embryo from a parent injected with purified mex-3B antisense RNA. Theseembryos and the parent animals retain the mex-3 mRNA, although levelsmay have been somewhat less than wild type. Panel D: Embryo from aparent injected with dsRNA corresponding to mex-3B; no mex-3 RNA wasdetected. Scale: each embryo is approximately 50 μm in length.

FIG. 4 shows inhibitory activity of unc-22A as a function of structureand concentration. The main graph indicates fractions in each behavioralclass. Embryos in the uterus and already covered with an eggshell at thetime of injection were not affected and, thus, are not included. Progenycohort groups are labeled 1 for 0-6 hours, 2 for 6-15 hours, 3 for 15-27hours, 4 for 27-41 hours, and 5 for 41-56 hours The bottom-left diagramshows genetically derived relationship between unc-22 gene dosage andbehavior based on analyses of unc-22 heterozygotes and polyploids^(8,3).

FIGS. 5 A-C show examples of genetic inhibition following ingestion byC. elegans of dsRNAs from expressing bacteria. Panel A: General strategyfor production of dsRNA by cloning a segment of interest betweenflanking copies of the bacteriophage T7 promoter and transcribing bothstrands of the segment by transfecting a bacterial strain (BL21/DE3)²⁸expressing the T7 polymerase gene from an inducible (Lac) promoter.Panel B: A GFP-expressing C. elegans strain, PD4251 (see FIG. 2), fed ona native bacterial host Panel C: PD4251 animals reared on a diet ofbacteria expressing dsRNA corresponding to the coding region for gfp.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of producing sequence-specificinhibition of gene expression by introducing double-stranded RNA(dsRNA). A process is provided for inhibiting expression of a targetgene in a cell. The process comprises introduction of RNA with partialor fully double-stranded character into the cell. Inhibition issequence-specific in that a nucleotide sequence from a portion of thetarget gene is chosen to produce inhibitory RNA. We disclose that thisprocess is (1) effective in producing inhibition of gene expression, (2)specific to the targeted gene, and (3) general in allowing inhibition ofmany different types of target gene.

The target gene may be a gene derived from the cell (i.e., a cellulargene), an endogenous gene (i.e., a cellular gene present in the genome),a transgene (i.e., a gene construct inserted at an ectopic site in thegenome of the cell), or a gene from a pathogen which is capable ofinfecting an organism from which the cell is derived. Depending on theparticular target gene and the dose of double stranded RNA materialdelivered, this process may provide partial or complete loss of functionfor the target gene. A reduction or loss of gene expression in at least99% of targeted cells has been shown.

Inhibition of gene expression refers to the absence (or observabledecrease) in the level of protein and/or mRNA product from a targetgene. Specificity refers to the ability to inhibit the target genewithout manifest effects on other genes of the cell. The consequences ofinhibition can be confirmed by examination of the outward properties ofthe cell or organism (as presented below in the examples) or bybiochemical techniques such as RNA solution hybridization, nucleaseprotection, Northern hybridization, reverse transcription, geneexpression monitoring with a microarray, antibody binding, enzyme linkedimmunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA),other immunoassays, and fluorescence activated cell analysis (FACS). ForRNA-mediated inhibition in a cell line or whole organism, geneexpression is conveniently assayed by use of a reporter or drugresistance gene whose protein product is easily assayed. Such reportergenes include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase(NOS), octopine synthase (OCS), and derivatives thereof. Multipleselectable markers are available that confer resistance to ampicillin,bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin,lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.

Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the present invention. Lower doses of injected material andlonger times after administration of dsRNA may result in inhibition in asmaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or95% of targeted cells). Quantitation of gene expression in a cell mayshow similar amounts of inhibition at the level of accumulation oftarget mRNA or translation of target protein. As an example, theefficiency of inhibition may be determined by assessing the amount ofgene product in the cell: mRNA may be detected with a hybridizationprobe having a nucleotide sequence outside the region used for theinhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

The RNA may comprise one or more strands of polymerized ribonucleotide.It may include modifications to either the phosphate-sugar backbone orthe nucleoside. For example, the phosphodiester linkages of natural RNAmay be modified to include at least one of a nitrogen or sulfurheteroatom. Modifications in RNA structure may be tailored to allowspecific genetic inhibition while avoiding a general panic response insome organisms which is generated by dsRNA. Likewise, bases may bemodified to block the activity of adenosine deaminase. RNA may beproduced enzymatically or by partial/total organic synthesis, anymodified ribonucleotide can be introduced by in vitro enzymatic ororganic synthesis.

The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. RNAduplex formation may be initiated either inside or outside the cell. TheRNA may be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of double-stranded material may yield more effectiveinhibition; lower doses may also be useful for specific applications.Inhibition is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition.

RNA containing a nucleotide sequences identical to a portion of thetarget gene are preferred for inhibition. RNA sequences with insertions,deletions, and single point mutations relative to the target sequencehave also been found to be effective for inhibition. Thus, sequenceidentity may optimized by sequence comparison and alignment algorithmsknown in the art (see Gribskov and Devereux, Sequence Analysis Primer,Stockton Press, 1991, and references cited therein) and calculating thepercent difference between the nucleotide sequences by, for example, theSmith-Waterman algorithm as implemented in the BESTFIT software programusing default parameters (e.g., University of Wisconsin GeneticComputing Group). Greater than 90% sequence identity, or even 100%sequence identity, between the inhibitory RNA and the portion of thetarget gene is preferred. Alternatively, the duplex region of the RNAmay be defined functionally as a nucleotide sequence that is capable ofhybridizing with a portion of the target gene transcript (e.g., 400 mMNaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for12-16 hours; followed by washing). The length of the identicalnucleotide sequences may be at least 25, 50, 100, 200, 300 or 400 bases.

As disclosed herein, 100% sequence identity between the RNA and thetarget gene is not required to practice the present invention. Thus theinvention has the advantage of being able to tolerate sequencevariations that might be expected due to genetic mutation, strainpolymorphism, or evolutionary divergence.

The cell with the target gene may be derived from or contained in anyorganism. The organism may a plant, animal, protozoan, bacterium, virus,or fungus. The plant may be a monocot, dicot or gymnosperm; the animalmay be a vertebrate or invertebrate. Preferred microbes are those usedin agriculture or by industry, and those that are pathogenic for plantsor animals. Fungi include organisms in both the mold and yeastmorphologies.

Plants include arabidopsis; field crops (e.g., alfalfa, barley, bean,corn, cotton, flax, pea, rape, rice, rye, safflower, sorghum, soybean,sunflower, tobacco, and wheat); vegetable crops (e.g., asparagus, beet,broccoli, cabbage, carrot, cauliflower, celery, cucumber, eggplant,lettuce, onion, pepper, potato, pumpkin, radish, spinach, squash, taro,tomato, and zucchini); fruit and nut crops (e.g., almond, apple,apricot, banana, blackberry, blueberry, cacao, cherry, coconut,cranberry, date, fajoa, filbert, grape, grapefruit, guava, kiwi, lemon,lime, mango, melon, nectarine, orange, papaya, passion fruit, peach,peanut, pear, pineapple, pistachio, plum, raspberry, strawberry,tangerine, walnut, and watermelon); and ornamentals (e.g., alder, ash,aspen, azalea, birch, boxwood, camellia, carnation, chrysanthemum, elm,fir, ivy, jasmine, juniper, oak, palm, poplar, pine, redwood,rhododendron, rose, and rubber).

Examples of vertebrate animals include fish, mammal, cattle, goat, pig,sheep, rodent, hamster, mouse, rat, primate, and human; invertebrateanimals include nematodes, other worms, drosophila, and other insects.Representative generae of nematodes include those that infect animals(e.g., Ancylostoma, Ascaridia, Ascaris, Bunostomum, Caenorhabditis,Capillaria, Chabertia, Cooperia, Dictyocaulus, Haemonchus, Heterakis,Nematodirus, Oesophagostomum, Ostertagia, Oxyuris, Parascaris,Strongylus, Toxascaris, Trichuris, Trichostrongylus, Tfhchonema,Toxocara, Uncinaria) and those that infect plants (e.g.,Bursaphalenchus, Criconemella, Diiylenchus, Ditylenchus, Globodera,Helicotylenchus, Heterodera, Longidorus, Melodoigyne, Nacobbus,Paratylenchus, Pratylenchus, Radopholus, Rotelynchus, Tylenchus, andXiphinema). Representative orders of insects include Coleoptera,Diptera, Lepidoptera, and Homoptera.

The cell having the target gene may be from the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, or the like. The cell may be astem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

RNA may be synthesized either in vivo or in vitro. Endogenous RNApolymerase of the cell may mediate transcription in vivo, or cloned RNApolymerase can be used for transcription in vivo or in vitro. Fortranscription from a transgene in vivo or an expression construct, aregulatory region (e.g., promoter, enhancer, silencer, splice donor andacceptor, polyadenylation) may be used to transcribe the RNA strand (orstrands). Inhibition may be targeted by specific transcription in anorgan, tissue, or cell type; stimulation of an environmental condition(e.g., infection, stress, temperature, chemical inducers); and/orengineering transcription at a developmental stage or age. The RNAstrands may or may not be polyadenylated; the RNA strands may or may notbe capable of being translated into a polypeptide by a cell'stranslational apparatus. RNA may be chemically or enzymaticallysynthesized by manual or automated reactions. The RNA may be synthesizedby a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g.,T3, T7, SP6). The use and production of an expression construct areknown in the art^(32, 33, 34) (see also WO 97/32016; U.S. Pat. Nos.5,593,874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693; and thereferences cited therein). If synthesized chemically or by in vitroenzymatic synthesis, the RNA may be purified prior to introduction intothe cell. For example, RNA can be purified from a mixture by extractionwith a solvent or resin, precipitation, electrophoresis, chromatography,or a combination thereof. Alternatively, the RNA may be used with no ora minimum of purification to avoid losses due to sample processing. TheRNA may be dried for storage or dissolved in an aqueous solution. Thesolution may contain buffers or salts to promote annealing, and/orstabilization of the duplex strands.

RNA may be directly introduced into the cell (i.e., intracellularly); orintroduced extracellularly into a cavity, interstitial space, into thecirculation of an organism, introduced orally, or may be introduced bybathing an organism in a solution containing the RNA. Methods for oralintroduction include direct mixing of the RNA with food of the organism,as well as engineered approaches in which a species that is used as foodis engineered to express the RNA, then fed to the organism to beaffected. For example, the RNA may be sprayed onto a plant or a plantmay be genetically engineered to express the RNA in an amount sufficientto kill some or all of a pathogen known to infect the plant. Physicalmethods of introducing nucleic acids, for example, injection directlyinto the cell or extracellular injection into the organism, may also beused. We disclose herein that in C. elegans, double-stranded RNAintroduced outside the cell inhibits gene expression. Vascular orextravascular circulation, the blood or lymph system, the phloem, theroots, and the cerebrospinal fluid are sites where the RNA may beintroduced. A transgenic organism that expresses RNA from a recombinantconstruct may be produced by introducing the construct into a zygote, anembryonic stem cell, or another multipotent cell derived from theappropriate organism.

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, promote annealing of the duplex strands,stabilize the annealed strands, or other-wise increase inhibition of thetarget gene.

The present invention may be used to introduce RNA into a cell for thetreatment or prevention of disease. For example, dsRNA may be introducedinto a cancerous cell or tumor and thereby inhibit gene expression of agene required for maintenance of the carcinogenic/tumorigenic phenotype.To prevent a disease or other pathology, a target gene may be selectedwhich is required for initiation or maintenance of thedisease/pathology. Treatment would include amelioration of any symptomassociated with the disease or clinical indication associated with thepathology.

A gene derived from any pathogen may be targeted for inhibition. Forexample, the gene could cause immunosuppression of the host directly orbe essential for replication of the pathogen, transmission of thepathogen, or maintenance of the infection. The inhibitory RNA could beintroduced in cells in vitro or ex vivo and then subsequently placedinto an animal to affect therapy, or directly treated by in vivoadministration. A method of gene therapy can be envisioned. For example,cells at risk for infection by a pathogen or already infected cells,particularly human immunodeficiency virus (HIV) infections, may betargeted for treatment by introduction of RNA according to theinvention. The target gene might be a pathogen or host gene responsiblefor entry of a pathogen into its host, drug metabolism by the pathogenor host, replication or integration of the pathogen's genome,establishment or spread of an infection in the host, or assembly, of thenext generation of pathogen. Methods of prophylaxis (i.e., prevention ordecreased risk of infection), as well as reduction in the frequency orseverity of symptoms associated with infection, can be envisioned.

The present invention could be used for treatment or development oftreatments for cancers of any type, including solid tumors andleukemias, including: apudoma, choristoma, branchioma, malignantcarcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker,basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ,Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell,papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, andtransitional cell), histiocytic disorders, leukemia (e.g., B cell, mixedcell, null cell, T cell, T-cell chronic, HTLV-II-associated, lymphocyticacute, lymphocytic chronic, mast cell, and myeloid), histiocytosismalignant, Hodgkin disease, immunoproliferative small, non-Hodgkinlymphoma, plasmacytoma, reticuloendotheliosis, melanoma,chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giantcell tumors, histiocytoma, lipoma, liposarcoma, mesothema, myxoma,myxosarcoma, osteoma, osteosarcoma, Ewing sarcoma, synovioma,adenofibroma, adenolymphoma, carcinosarcoma, chordoma,cranio-pharyngioma, dysgerminoma, hamartoma, mesenchymoma, mesonephroma,myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma,trophoblastic tumor, adenocarcinoma, adenoma, cholangioma,cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosacell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor,Leydig cell tumor, papilloma, Sertoli cell tumor, theca cell tumor,leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma,rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma,meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma,neuroma, paraganglioma, paraganglioma nonchromaffin, angiokeratoma,angiolymphoid hyperplasia with eosinophilia, angioma sclerosing,angiomatosis, glomangioma, hemangioendothelioma, hemangioma,hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma,lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma,cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma,leukosarcoma, liposarcomi, lymphangiosarcoma, myosarcoma, myxosarcoma,ovarian carcinoma, rhabdomyosarcoma, sarcoma (e.g., Ewing, experimental,Kaposi, and mast cell), neoplasms (e.g., bone, breast, digestive system,colorectal, liver, pancreatic, pituitary, testicular, orbital, head andneck, central nervous system, acoustic, pelvic, respiratory tract, andurogenital), neurofibromatosis, and cervical dysplasia, and fortreatment of other conditions in which cells have become immortalized ortransformed. The invention could be used in combination with othertreatment modalities, such as chemotherapy, cryotherapy, hyperthermia,radiation therapy, and the like.

As disclosed herein, the present invention may is not limited to anytype of target gene or nucleotide sequence. But the following classes ofpossible target genes are listed for illustrative purposes:developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors,Wnt family members, Pax family members, Winged helix family members, Hoxfamily members, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors); oncogenes (e.g., ABL1, BCL1, BCL2, BCL6, CBFA2,CBL, CSF1R, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR,HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1,PML, RET, SRC, TAL1, TCL3, and YES); tumor suppressor genes (e.g., APC,BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53, and WT1); and enzymes(e.g., ACC synthases and oxidases, ACP desaturases and hydroxylases,ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases,amyloglucosidases, catalases, cellulases, chalcone synthases,chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and RNApolymerases, galactosidases, glucanases, glucose oxidases, granule-boundstarch synthases, GTPases, helicases, hemicellulases, integrases,inulinases, invertases, isomerases, kinases, lactases, lipases,lipoxygenases, lysozymes, nopaline synthases, octopine synthases,pectinesterases, peroxidases, phosphatases, phospholipases,phosphorylases, phytases, plant growth regulator synthases,polygalacturonases, proteinases and peptidases, pullanases,recombinases, reverse transcriptases, RUBISCOS, topoisomerases, andxylanases).

The present invention could comprise a method for producing plants withreduced susceptibility to climatic injury, susceptibility to insectdamage, susceptibility to infection by a pathogen, or altered fruitripening characteristics. The targeted gene may be an enzyme, a plantstructural protein, a gene involved in pathogenesis, or an enzyme thatis involved in the production of a non-proteinaceous part of the plant(i.e., a carbohydrate or lipid). If an expression construct is used totranscribe the RNA in a plant, transcription by a wound- orstress-inducible; tissue-specific (e.g., fruit, seed, anther, flower,leaf, root); or otherwise regulatable (e.g., infection, light,temperature, chemical) promoter may be used. By inhibiting enzymes atone or more points in a metabolic pathway or genes involved inpathogenesis, the effect may be enhanced: each activity will be affectedand the effects may be magnified by targeting multiple differentcomponents. Metabolism may also be manipulated by inhibiting feedbackcontrol in the pathway or production of unwanted metabolic byproducts.

The present invention may be used to reduce crop destruction by otherplant pathogens such as arachnids, insects, nematodes, protozoans,bacteria, or fungi. Some such plants and their pathogens are listed inIndex of Plant Diseases in the United States (U.S. Dept. of AgricultureHandbook No. 165, 1960); Distribution of Plant-Parasitic NematodeSpecies in North America (Society of Nematologists, 1985); and Fungi onPlants and Plant Products in the United States (AmericanPhytopathological Society, 1989). Insects with reduced ability to damagecrops or improved ability to prevent other destructive insects fromdamaging crops may be produced. Furthermore, some nematodes are vectorsof plant pathogens, and may be attacked by other beneficial nematodeswhich have no effect on plants. Inhibition of target gene activity couldbe used to delay or prevent entry into a particular developmental step(e.g., metamorphosis), if plant disease was associated with a particularstage of the pathogen's life cycle. Interactions between pathogens mayalso be modified by the invention to limit crop damage. For example, theability of beneficial nematodes to attack their harmful prey may beenhanced by inhibition of behavior-controlling nematode genes accordingto the invention.

Although pathogens cause disease, some of the microbes interact withtheir plant host in a beneficial manner. For example, some bacteria areinvolved in symbiotic relationships that fix nitrogen and some fungiproduce phytohormones. Such beneficial interactions may be promoted byusing the present invention to inhibit target gene activity in the plantand/or the microbe.

Another utility of the present invention could be a method ofidentifying gene function in an organism comprising the use ofdouble-stranded RNA to inhibit the activity of a target gene ofpreviously unknown function. Instead of the time consuming and laboriousisolation of mutants by traditional genetic screening, functionalgenomics would envision determining the function of uncharacterizedgenes by employing the invention to reduce the amount and/or alter thetiming of target gene activity. The invention could be used indetermining potential targets for pharmaceutics, understanding normaland pathological events associated with development, determiningsignaling pathways responsible for postnatal development/aging, and thelike. The increasing speed of acquiring nucleotide sequence informationfrom genomic and expressed gene sources, including total sequences forthe yeast, D. melanogaster, and C. elegans genomes, can be coupled withthe invention to determine gene function in an organism (e.g.,nematode). The preference of different organisms to use particularcodons, searching sequence databases for related gene products,correlating the linkage map of genetic traits with the physical map fromwhich the nucleotide sequences are derived, and artificial intelligencemethods may be used to define putative open reading frames from thenucleotide sequences acquired in such sequencing projects.

A simple assay would be to inhibit gene expression according to thepartial sequence available from an expressed sequence tag (EST).Functional alterations in growth, development, metabolism, diseaseresistance, or other biological processes would be indicative of thenormal role of the EST's gene product.

The ease with which RNA can be introduced into an intact cell/organismcontaining the target gene allows the present invention to be used inhigh throughput screening (HTS). For example, duplex RNA can be producedby an amplification reaction using primers flanking the inserts of anygene library derived from the target cell/organism. Inserts may bederived from genomic DNA or mRNA (e.g., cDNA and cRNA). Individualclones from the library can be replicated and then isolated in separatereactions, but preferably the library is maintained in individualreaction vessels (e.g., a 96-well microtiter plate) to minimize thenumber of steps required to practice the invention and to allowautomation of the process. Solutions containing duplex RNAs that arecapable of inhibiting the different expressed genes can be placed intoindividual wells positioned on a microtiter plate as an ordered array,and intact cells/organisms in each well can be assayed for any changesor modifications in behavior or development due to inhibition of targetgene activity. The amplified RNA can be fed directly to, injected into,the cell/organism containing the target gene. Alternatively, the duplexRNA can be produced by in vivo or in vitro transcription from anexpression construct used to produce the library. The construct can bereplicated as individual clones of the library and transcribed toproduce the RNA; each clone can then be fed to, or injected into, thecell/organism containing the target gene. The function of the targetgene can be assayed from the effects it has on the cell/organism whengene activity is inhibited. This screening could be amenable to smallsubjects that can be processed in large number, for example:arabidopsis, bacteria, drosophila, fungi, nematodes, viruses, zebrafish,and tissue culture cells derived from mammals.

A nematode or other organism that produces a colorimetric, fluorogenic,or luminescent signal in response to a regulated promoter (e.g.,transfected with a reporter gene construct) can be assayed in an HTSformat to identify DNA-binding proteins that regulate the promoter. Inthe assay's simplest form, inhibition of a negative regulator results inan increase of the signal and inhibition of a positive regulator resultsin a decrease of the signal.

If a characteristic of an organism is determined to be geneticallylinked to a polymorphism through RFLP or QTL analysis, the presentinvention can be used to gain insight regarding whether that geneticpolymorphism might be directly responsible for the characteristic. Forexample, a fragment defining the genetic polymorphism or sequences inthe vicinity of such a genetic polymorphism can be amplified to producean RNA, the duplex RNA can be introduced to the organism, and whether analteration in the characteristic is correlated with inhibition can bedetermined. Of course, there may be trivial explanations for negativeresults with this type of assay, for example: inhibition of the targetgene causes lethality, inhibition of the target gene may not result inany observable alteration, the fragment contains nucleotide sequencesthat are not capable of inhibiting the target gene, or the target gene'sactivity is redundant.

The present invention may be useful in allowing the inhibition ofessential genes. Such genes may be required for cell or organismviability at only particular stages of development or cellularcompartments. The functional equivalent of conditional mutations may beproduced by inhibiting activity of the target gene when or where it isnot required for viability. The invention allows addition of RNA atspecific times of development and locations in the organism withoutintroducing permanent mutations into the target genome.

If alternative splicing produced a family of transcripts that weredistinguished by usage of characteristic exons, the present inventioncan target inhibition through the appropriate exons to specificallyinhibit or to distinguish among the functions of family members. Forexample, a hormone that contained an alternatively spliced transmembranedomain may be expressed in both membrane bound and secreted forms.Instead of isolating a nonsense mutation that terminates translationbefore the transmembrane domain, the functional consequences of havingonly secreted hormone can be determined according to the invention bytargeting the exon containing the transmembrane domain and therebyinhibiting expression of membrane-bound hormone.

The present invention may be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples or subjects. Preferredcomponents are the dsRNA and a vehicle that promotes introduction of thedsRNA. Such a kit may also include instructions to allow a user of thekit to practice the invention.

Pesticides may include the RNA molecule itself, an expression constructcapable of expressing the RNA, or organisms transfected with theexpression construct. The pesticide of the present invention may serveas an arachnicide, insecticide, nematicide, viricide, bactericide,and/or fungicide. For example, plant parts that are accessible aboveground (e.g., flowers, fruits, buds, leaves, seeds, shoots, bark, stems)may be sprayed with pesticide, the soil may be soaked with pesticide toaccess plant parts growing beneath ground level, or the pest may becontacted with pesticide directly. If pests interact with each other,the RNA may be transmitted between them. Alternatively, if inhibition ofthe target gene results in a beneficial effect on plant growth ordevelopment, the aforementioned RNA, expression construct, ortransfected organism may be considered a nutritional agent. In eithercase, genetic engineering of the plant is not required to achieve theobjectives of the invention.

Alternatively, an organism may be engineered to produce dsRNA whichproduces commercially or medically beneficial results, for example,resistance to a pathogen or its pathogenic effects, improved growth, ornovel developmental patterns.

Used as either an pesticide or nutrient, a formulation of the presentinvention may be delivered to the end user in dry or liquid form: forexample, as a dust, granulate, emulsion, paste, solution, concentrate,suspension, or encapsulation. Instructions for safe and effective usemay also be provided with the formulation. The formulation might be useddirectly, but concentrates would require dilution by mixing with anextender provided by the formulator or the end user. Similarly, anemulsion, paste, or suspension may require the end user to performcertain preparation steps before application. The formulation mayinclude a combination of chemical additives known in the art such assolid carriers, minerals, solvents, dispersants, surfactants,emulsifiers, tackifiers, binders, and other adjuvants. Preservatives andstabilizers may also be added to the formulation to facilitate storage.The crop area or plant may also be treated simultaneously or separatelywith other pesticides or fertilizers. Methods of application includedusting, scattering or pouring, soaking, spraying, atomizing, andcoating. The precise physical form and chemical composition of theformulation, and its method of application, would be chosen to promotethe objectives of the invention and in accordance with prevailingcircumstances. Expression constructs and transfected hosts capable ofreplication may also promote the persistence and/or spread of theformulation.

Description of the dsRNA Inhibition Phenomenon in C. elegans

The operation of the present invention was shown in the model geneticorganism Caenorhabditis elegans.

Introduction of RNA into cells had been seen in certain biologicalsystems to interfere with function of an endogenous gene^(1,2). Manysuch effects were believed to result from a simple antisense mechanismdependent on hybridization between injected single-stranded RNA andendogenous transcripts. In other cases, a more complex mechanism hadbeen suggested. One instance of an RNA-mediated mechanism was RNAinterference (RNAi) phenomenon in the nematode C. elegans. RNAi had beenused in a variety of studies to manipulate gene expression^(3,4).

Despite the usefulness of RNAi in C. elegans, many features had beendifficult to explain. Also, the lack of a clear understanding of thecritical requirements for interfering RNA led to a sporadic record offailure and partial success in attempts to extend RNAi beyond theearliest stages following injection. A statement frequently made in theliterature was that sense and antisense RNA preparations are eachsufficient to cause interference^(3,4). The only precedent for such asituation was in plants where the process of cosuppression had a similarhistory of usefulness in certain cases, failure in others, and noability to design interference protocols with a high chance of success.Working with C. elegans, we discovered an RNA structure that would giveeffective and uniform genetic inhibition. The prior art did not teach orsuggest that RNA structure was a critical feature for inhibition of geneexpression. Indeed the ability of crude sense and antisense preparationsto produce interference^(3,4) had been taken as an indication that RNAstructure was not a critical factor. Instead, the extensive plantliterature and much of the ongoing research in C. elegans was focused onthe possibility that detailed features of the target gene sequence orits chromosomal locale was the critical feature for interfering withgene expression.

The inventors carefully purified sense or antisense RNA for unc-22 andtested each for gene-specific inhibition. While the crude sense andantisense preparations had strong interfering activity, it was foundthat the purified sense and antisense RNAs had only marginal inhibitoryactivity. This was unexpected because many techniques in molecularbiology are based on the assumption that RNA produced with specific invitro promoters (e.g., T3 or T7 RNA polymerase), or with characterizedpromoters in vivo, is produced predominantly from a single strand. Theinventors had carried out purification of these crude preparations toinvestigate whether a small fraction of the RNA had an unusual structurewhich might be responsible for the observed genetic inhibition. Torigorously test whether double-stranded character might contribute togenetic inhibition, the inventors carried out additional purification ofsingle-stranded RNAs and compared inhibitory activities of individualstrands with that of the double-stranded hybrid.

The following examples are meant to be illustrative of the presentinvention; however, the practice of the invention is not limited orrestricted in any way by them.

Analysis of RNA-Mediated Inhibition of C. elegans Genes

The unc-22 gene was chosen for initial comparisons of activity as aresult of previous genetic analysis that yields a semi-quantitativecomparison between unc-22 gene activity and the movement phenotypes ofanimals^(3,8): decreases in activity produce an increasingly severetwitching phenotype, while complete loss of function results in theadditional appearance of muscle structural defects and impairedmotility. unc-22 encodes an abundant but non-essential myofilamentprotein⁷⁻⁹. unc-22 mRNA is present at several thousand copies perstriated muscle cell³.

Purified antisense and sense RNAs covering a 742 nt segment of unc-22had only marginal inhibitory activity, requiring a very high dose ofinjected RNA for any observable effect (FIG. 4). By contrast, asense+antisense mixture produced a highly effective inhibition ofendogenous gene activity (FIG. 4). The mixture was at least two ordersof magnitude more effective than either single strand in inhibiting geneexpression. The lowest dose of the sense+antisense mixture tested,approximately 60,000 molecules of each strand per adult, led totwitching phenotypes in an average of 100 progeny. unc-22 expressionbegins in embryos with approximately 500 cells. At this point, theoriginal injected material would be diluted to at most a few moleculesper cell.

The potent inhibitory activity of the sense+antisense mixture couldreflect formation of double-stranded RNA (dsRNA), or conceivably somealternate synergy between the strands. Electrophoretic analysisindicated that the injected material was predominantly double stranded.The dsRNA was gel purified from the annealed mixture and found to retainpotent inhibitory activity. Although annealing prior to injection wascompatible with inhibition, it was not necessary. Mixing of sense andantisense RNAs in low salt (under conditions of minimal dsRNAformation), or rapid sequential injection of sense and antisensestrands, were sufficient to allow complete inhibition. A long interval(>1 hour) between sequential injections of sense and antisense RNAresulted in a dramatic decrease in inhibitory activity. This suggeststhat injected single strands may be degraded or otherwise renderedinaccessible in the absence of the complementary strand.

An issue of specificity arises when considering known cellular responsesto dsRNA. Some organisms have a dsRNA-dependent protein kinase thatactivates a panic response mechanism¹⁰. Conceivably, the inventivesense+antisense synergy could reflect a non-specific potentiation ofantisense effects by such a panic mechanism. This was not found to bethe case: co-injection of dsRNA segments unrelated to unc-22 did notpotentiate the ability of unc-22 single strands to mediate inhibition.Also investigated was whether double-stranded structure could potentiateinhibitory activity when placed in cis to a single-stranded segment. Nosuch potentiation was seen; unrelated double-stranded sequences located5′ or 3′ of a single-stranded unc-22 segment did not stimulateinhibition. Thus potentiation of gene-specific inhibition was observedonly when dsRNA sequences exist within the region of homology with thetarget gene.

The phenotype produced by unc-22 dsRNA was specific. Progeny of injectedanimals exhibited behavior indistinguishable from characteristic unc-22loss of function mutants. Target-specificity of dsRNA effects usingthree additional genes with well characterized phenotypes (FIG. 1 andTable 1). unc-54 encodes a body wall muscle myosin heavy chain isoformrequired for full muscle contraction^(7,11,12), fem-1 encodes anankyrin-repeat containing protein required in hermaphrodites for spermproduction^(13,14), and hlh-1 encodes a C. elegans homolog of the myoDfamily required for proper body shape and motility^(15,16). For each ofthese genes, injection of dsRNA produced progeny broods exhibiting theknown null mutant phenotype, while the purified single strands producedno significant reduction in gene expression. With one exception, all ofthe phenotypic consequences of dsRNA injection were those expected frominhibition of the corresponding gene. The exception (segment unc54C,which led to an embryonic and larval arrest phenotype not seen withunc-54 null mutants) was illustrative. This segment covers the highlyconserved myosin motor domain, and might have been expected to inhibitthe activity of other highly related myosin heavy chain genes¹⁷. Thisinterpretation would support uses of the present invention in whichnucleotide sequence comparison of dsRNA and target gene show less than100% identity. The unc54C segment has been unique in our overallexperience to date: effects of 18 other dsRNA segments have all beenlimited to those expected from characterized null mutants.

The strong phenotypes seen following dsRNA injection are indicative ofinhibitory effects occurring in a high fraction of cells. The unc-54 andhlh-1 muscle phenotypes, in particular, are known to result from a largenumber of defective muscle cells^(11,16). To examine inhibitory effectsof dsRNA on a cellular level, a transgenic line expressing two differentGFP-derived fluorescent reporter proteins in body muscle was used.Injection of dsRNA directed to gfp produced dramatic decreases in thefraction of fluorescent cells (FIG. 2). Both reporter proteins wereabsent from the negative cells, while the few positive cells generallyexpressed both GFP forms.

The pattern of mosaicism observed with gfp inhibition was not random. Atlow doses of dsRNA, the inventors saw frequent inhibition in theembryonically-derived muscle cells present when the animal hatched. Theinhibitory effect in these differentiated cells persisted through larvalgrowth: these cells produced little or no additional GFP as the affectedanimals grew. The 14 postembryonically-derived striated muscles are bornduring early larval stages and were more resistant to inhibition. Thesecells have come through additional divisions (13-14 versus 8-9 forembryonic muscles^(18,19)). At high concentrations of gfp dsRNA,inhibition was noted in virtually all striated bodywall muscles, withoccasional single escaping cells including cells born in embryonic orpostembryonic stages. The nonstriated vulval muscles, born during latelarval development, appeared resistant to genetic inhibition at alltested concentrations of injected RNA. The latter result is importantfor evaluating the use of the present invention in other systems. First,it indicates that failure in one set of cells from an organism does notnecessarily indicate complete non-applicability of the invention to thatorganism. Second, it is important to realize that not all tissues in theorganism need to be affected for the invention to be used in anorganism. This may serve as an advantage in some situations.

A few observations serve to clarify the nature of possible targets andmechanisms for RNA mediated genetic inhibition in C. elegans:

First, dsRNA segments corresponding to a variety of intron and promotersequences did not produce detectable inhibition (Table 1). Althoughconsistent with possible inhibition at a post-transcriptional level,these experiments do not rule out inhibition at the level of the gene.

Second, dsRNA injection produced a dramatic decrease in the level of theendogenous mRNA transcript (FIG. 3). Here, a mex-3 transcript that isabundant in the gonad and early embryos²⁰ was targeted, wherestraightforward in situ hybridization can be performed⁵. No endogenousmex-3 mRNA was observed in animals injected with a dsRNA segment derivedfrom mex-3 (FIG. 3D), but injection of purified mex-3 antisense RNAresulted in animals that retained substantial endogenous mRNA levels(FIG. 3C).

Third, dsRNA-mediated inhibition showed a surprising ability to crosscellular boundaries. Injection of dsRNA for unc-22, gfp, or lacZ intothe body cavity of the head or tail produced a specific and robustinhibition of gene expression in the progeny brood (Table 2). Inhibitionwas seen in the progeny of both gonad arms, ruling out a transient“nicking” of the gonad in these injections. dsRNA injected into bodycavity or gonad of young adults also produced gene-specific inhibitionin somatic tissues of the injected animal (Table 2).

Table 3 shows that C. elegans can respond in a gene-specific manner todsRNA encountered in the environment. Bacteria are a natural food sourcefor C. elegans. The bacteria are ingested, ground in the animal'spharynx, and the bacterial contents taken up in the gut. The resultsshow that E. coli bacteria expressing dsRNAs can confer specificinhibitory effects on C. elegans nematode larvae that feed on them.

Three C. elegans genes were analyzed. For each gene, corresponding dsRNAwas expressed in E. coli by inserting a segment of the coding regioninto a plasmid construct designed for bidirectional transcription bybacteriophage T7 RNA polymerase. The dsRNA segments used for theseexperiments were the same as those used in previous microinjectionexperiments (see FIG. 1). The effects resulting from feeding thesebacteria to C. elegans were compared to the effects achieved bymicroinjecting animals with dsRNA.

The C. elegans gene unc-22 encodes an abundant muscle filament protein.unc-22 null mutations produce a characteristic and uniform twitchingphenotype in which the animals can sustain only transient musclecontraction. When wild-type animals were fed bacteria expressing a dsRNAsegment from unc-22, a high fraction (85%) exhibited a weak but stilldistinct twitching phenotype characteristic of partial loss of functionfor the unc-22 gene. The C. elegans fem-1 gene encodes a late componentof the sex determination pathway. Null mutations prevent the productionof sperm and lead euploid (XX) animals to develop as females, while wildtype XX animals develop as hermaphrodites. When wild-type animals werefed bacteria expressing dsRNA corresponding to fem-1, a fraction (43%)exhibit a sperm-less (female) phenotype and were sterile. Finally, theability to inhibit gene expression of a transgene target was assessed.When animals carrying a gfp transgene were fed bacteria expressing dsRNAcorresponding to the gfp reporter, an obvious decrease in the overalllevel of GFP fluorescence was observed, again in approximately 12% ofthe population (see FIG. 5, panels B and C).

The effects of these ingested RNAs were specific. Bacteria carryingdifferent dsRNAs from fem-1 and gfp produced no twitching, dsRNAs fromunc-22 and fem-1 did not reduce gfp expression, and dsRNAs from gfp andunc-22 did not produce females. These inhibitory effects were apparentlymediated by dsRNA: bacteria expressing only the sense or antisensestrand for either gfp or unc-22 caused no evident phenotypic effects ontheir C. elegans predators.

Table 4 shows the effects of bathing C. elegans in a solution containingdsRNA. Larvae were bathed for 24 hours in solutions of the indicateddsRNAs (1 mg/ml), then allowed to recover in normal media and allowed togrow under standard conditions for two days. The unc-22 dsRNA wassegment ds-unc22A from FIG. 1. pos-1 and sqt-3 dsRNAs were from the fulllength cDNA clones. pos-1 encodes an essential maternally providedcomponent required early in embryogenesis. Mutations removing pos-1activity have an early embryonic arrest characteristic of skn-likemutations^(29,30). Cloning and activity patterns for sqt-3 have beendescribed³¹ . C. elegans sqt-3 mutants have mutations in the col-1collagen gene³¹. Phenotypes of affected animals are noted. Incidences ofclear phenotypic effects in these experiments were 5-10% for unc-22, 50%for pos-1, and 5% for sqt-3. These are frequencies of unambiguousphenocopies; other treated animals may have had marginal defectscorresponding to the target gene that were not observable. Eachtreatment was fully gene-specific in that unc-22 dsRNA produced onlyUnc-22 phenotypes, pos-1 dsRNA produced only Pos-1 phenotypes, and sqt-3dsRNA produced only Sqt-3 phenotypes.

Some of the results described herein were published after the filing ofour provisional application. Those publications and a review can becited as Fire, A., et al. Nature, 391, 806-811, 1998; Timmons, L. &Fire, A. Nature, 395, 854, 1998; and Montgomery, M. K. & Fire, A. Trendsin Genetics, 14, 255-258, 1998.

The effects described herein significantly augment available tools forstudying gene function in C. elegans and other organisms. In particular,functional analysis should now be possible for a large number ofinteresting coding regions²¹ for which no specific function have beendefined. Several of these observations show the properties of dsRNA thatmay affect the design of processes for inhibition of gene expression.For example, one case was observed in which a nucleotide sequence sharedbetween several myosin genes may inhibit gene expression of severalmembers of a related gene family.

Methods of RNA Synthesis and Microinjection

RNA was synthesized from phagemid clones with T3 and T7 RNA polymerase⁶,followed by template removal with two sequential DNase treatments. Incases where sense, antisense, and mixed RNA populations were to becompared, RNAs were further purified by electrophoresis onlow-gelling-temperature agarose. Gel-purified products appeared to lackmany of the minor bands seen in the original “sense” and “antisense”preparations. Nonetheless, RNA species accounting for less than 10% ofpurified RNA preparations would not have been observed. Without gelpurification, the “sense” and “antisense” preparations producedsignificant inhibition. This inhibitory activity was reduced oreliminated upon gel purification. By contrast, sense+antisense mixturesof gel purified and non-gel-purified RNA preparations produced identicaleffects.

Following a short (5 minute) treatment at 68° C. to remove secondarystructure, sense+antisense annealing was carried out in injectionbuffer²⁷ at 37° C. for 10-30 minutes. Formation of predominantly doublestranded material was confirmed by testing migration on a standard(non-denaturing) agarose gel: for each RNA pair, gel mobility wasshifted to that expected for double-stranded RNA of the appropriatelength. Co-incubation of the two strands in a low-salt buffer (5 mMTris-HCl pH 7.5, 0.5 mM EDTA) was insufficient for visible formation ofdouble-stranded RNA in vitro. Non-annealed sense+antisense RNAs forunc22B and gfpG were tested for inhibitory effect and found to be muchmore active than the individual single strands, but 2-4 fold less activethan equivalent pre-annealed preparations.

After pre-annealing of the single strands for unc22A, the singleelectrophoretic species corresponding in size to that expected for dsRNAwas purified using two rounds of gel electrophoresis. This materialretained a high degree of inhibitory activity.

Except where noted, injection mixes were constructed so animals wouldreceive an average of 0.5×10⁶ to 1.0×10⁶ molecules of RNA. Forcomparisons of sense, antisense, and dsRNA activities, injections werecompared with equal masses of RNA (i.e., dsRNA at half the molarconcentration of the single strands). Numbers of molecules injected peradult are given as rough approximations based on concentration of RNA inthe injected material (estimated from ethidium bromide staining) andinjection volume (estimated from visible displacement at the site ofinjection). A variability of several-fold in injection volume betweenindividual animals is possible; however, such variability would notaffect any of the conclusions drawn herein.

Methods for Analysis of Phenotypes

Inhibition of endogenous genes was generally assayed in a wild typegenetic background (N2). Features analyzed included movement, feeding,hatching, body shape, sexual identity, and fertility. Inhibition withgfp²⁷ and lacZ activity was assessed using strain PD4251. This strain isa stable transgenic strain containing an integrated array (ccIs4251)made up of three plasmids: pSAK4 (myo-3 promoter driving mitochondriallytargeted GFP), pSAK2 (myo-3 promoter driving a nuclear targeted GFP-LacZfusion), and a dpy-20 subclone²⁶ as a selectable marker. This strainproduces GFP in all body muscles, with a combination of mitochondrialand nuclear localization. The two distinct compartments are easilydistinguished in these cells, allowing a facile distinction betweencells expressing both, either, or neither of the original GFPconstructs.

Gonadal injection was performed by inserting the microinjection needleinto the gonadal syncitium of adults and expelling 20-100 pl of solution(see Reference 25). Body cavity injections followed a similar procedure,with needle insertion into regions of the head and tail beyond thepositions of the two gonad arms. Injection into the cytoplasm ofintestinal cells was another effective means of RNA delivery, and may bethe least disruptive to the animal. After recovery and transfer tostandard solid media, injected animals were transferred to fresh cultureplates at 16 hour intervals. This yields a series of semi-synchronouscohorts in which it was straightforward to identify phenotypicdifferences. A characteristic temporal pattern of phenotypic severity isobserved among progeny. First, there is a short “clearance” interval inwhich unaffected progeny are produced. These include impermeablefertilized eggs present at the time of injection. After the clearanceperiod, individuals are produced which show the inhibitory phenotype.After injected animals have produced eggs for several days, gonads canin some cases “revert” to produce incompletely affected orphenotypically normal progeny.

Additional Description of the Results

FIG. 1 shows genes used to study RNA-mediated genetic inhibition in C.elegans. Intron-exon structure for genes used to test RNA-mediatedinhibition are shown (exons: filled boxes; introns: open boxes; 5′ and3′ untranslated regions: shaded; sequence references are as follows:unc-22⁹, unc-54¹², fem-1¹⁴, and hlh-1¹⁵). These genes were chosen basedon: (1) a defined molecular structure, (2) classical genetic datashowing the nature of the null phenotype. Each segment tested forinhibitory effects is designated with the name of the gene followed by asingle letter (e.g., unc22C). Segments derived from genomic DNA areshown above the gene, segments derived from cDNA are shown below thegene. The consequences of injecting double-stranded RNA segments foreach of these genes is described in Table 1. dsRNA sequences from thecoding region of each gene produced a phenotype resembling the nullphenotype for that gene.

The effects of inhibitory RNA were analyzed in individual cells (FIG. 2,panels A-H). These experiments were carried out in a reporter strain(called PD4251) expressing two different reporter proteins: nuclearGFP-LacZ and mitochondrial GFP, both expressed in body muscle. Thefluorescent nature of these reporter proteins allowed us to examineindividual cells under the fluorescence microscope to determine theextent and generality of the observed inhibition of gene. ds-unc22A RNAwas injected as a negative control. GFP expression in progeny of theseinjected animals was not affected. The GFP patterns of these progenyappeared identical to the parent strain, with prominent fluorescence innuclei (the nuclear localized GFP-LacZ) and mitochondria (themitochondrially targeted GFP): young larva (FIG. 2A), adult (FIG. 2B),and adult body wall at high magnification (FIG. 2C).

In contrast, the progeny of animals injected with ds-gfpG RNA areaffected (FIGS. 2D-F). Observable GFP fluorescence is completely absentin over 95% of the cells. Few active cells were seen in larvae (FIG. 2Dshows a larva with one active cell; uninjected controls show GFPactivity in all 81 body wall muscle cells). Inhibition was not effectivein all tissues: the entire vulval musculature expressed active GFP in anadult animal (FIG. 2E). Rare GFP positive body wall muscle cells werealso seen adult animals (two active cells are shown in FIG. 2F).Inhibition was target specific (FIGS. 2G-I). Animals were injected withds-lacZL RNA, which should affect the nuclear but not the mitochondrialreporter construct. In the animals derived from this injection,mitochondrial-targeted GFP appeared unaffected while thenuclear-targeted GFP-LacZ was absent from almost all cells (larva inFIG. 2G). A typical adult lacked nuclear GFP-LacZ in almost allbody-wall muscles but retained activity in vulval muscles (FIG. 2H).Scale bars in FIG. 2 are 20 μm.

The effects of double-stranded RNA corresponding to mex-3 on levels ofthe endogenous mRNA was shown by in situ hybridization to embryos (FIG.3, panels AD). The 1262 nt mex-3 cDNA clone²⁰ was divided into twosegments, mex-3A and mex-3B with a short (325 nt) overlap. Similarresults were obtained in experiments with no overlap between inhibitingand probe segments. mex-3B antisense or dsRNA was injected into thegonads of adult animals, which were maintained under standard cultureconditions for 24 hours before fixation and in situ hybridization (seeReference 5). The mex-3B dsRNA produced 100% embryonic arrest,while >90% of embryos from the antisense injections hatched. Antisenseprobes corresponding to mex-3A were used to assay distribution of theendogenous mex-3 mRNA (dark stain). Four-cell stage embryos wereassayed; similar results were observed from the 1 to 8 cell stage and inthe germline of injected adults. The negative control (the absence ofhybridization probe) showed a lack of staining (FIG. 3A). Embryos fromuninjected parents showed a normal pattern of endogenous mex-3 RNA (FIG.3B). The observed pattern of mex-3 RNA was as previously described inReference 20. Injection of purified mex-3B antisense RNA produced atmost a modest effect: the resulting embryos retained mex-3 mRNA,although levels may have been somewhat less than wild type (FIG. 3C). Incontrast, no mex-3 RNA was detected in embryos from parents injectedwith dsRNA corresponding to mex-3B (FIG. 3D). The scale of FIG. 3 issuch that each embryo is approximately 50 μm in length.

Gene-specific inhibitory activity by unc-22A RNA was measured as afunction of RNA structure and concentration (FIG. 4). Purified antisenseand sense RNA from unc22A were injected individually or as an annealedmixture. “Control” was an unrelated dsRNA (gfpG). Injected animals weretransferred to fresh culture plates 6 hours (columns labeled 1), 15hours (columns labeled 2), 27 hours (columns labeled 3), 41 hours(columns labeled 4), and 56 hours (columns labeled 5) after injection.Progeny grown to adulthood were scored for movement in their growthenvironment, then examined in 0.5 mM levamisole. The main graphindicates fractions in each behavioral class. Embryos in the uterus andalready covered with an eggshell at the time of injection were notaffected and, thus, are not included in the graph. The bottom-leftdiagram shows the genetically derived relationship between unc-22 genedosage and behavior based on analyses of unc-22 heterozygotes andpolyploids^(8,3).

FIGS. 5 A-C show a process and examples of genetic inhibition followingingestion by C. elegans of dsRNAs from expressing bacteria. A generalstrategy for production of dsRNA is to clone segments of interestbetween flanking copies of the bacteriophage T7 promoter into abacterial plasmid construct (FIG. 5A). A bacterial strain (BL21/DE3)²⁸expressing the T7 polymerase gene from an inducible (Lac) promoter wasused as a host. A nuclease-resistant dsRNA was detected in lysates oftransfected bacteria. Comparable inhibition results were obtained withthe two bacterial expression systems. A GFP-expressing C. elegansstrain, PD4251 (see FIG. 2), was fed on a native bacterial host. Theseanimals show a uniformly high level of GFP fluorescence in body muscles(FIG. 5B). PD4251 animals were also reared on a diet of bacteriaexpressing dsRNA corresponding to the coding region for gfp. Under theconditions of this experiment, 12% of these animals showed dramaticdecreases in GFP (FIG. 5C). As an alternative strategy, single copies ofthe T7 promoter were used to drive expression of an inverted-duplicationfor a segment of the target gene, either unc-22 or gfp. This wascomparably effective.

All references (e.g., books, articles, applications, and patents) citedin this specification are indicative of the level of skill in the artand their disclosures are incorporated herein in their entirety.

-   1. Izant, J. & Weintraub, H. Cell 36, 1007-1015 (1984).-   2. Nellen, W. & Lichtenstein, C. TIBS 18, 419-423 (1993).-   3. Fire, A., et al. Development 113, 503-514 (1991).-   4. Guo, S. & Kemphues, K. Cell 81, 611-620 (1995).-   5. Seydoux, G. & Fire, A. Development 120, 2823-2834 (1994).-   6. Ausubel, F., et al. Current Protocols in Molecular Biology, John    Wiley N.Y. (1990).-   7. Brenner, S. Genetics 77, 71-94 (1974).-   8. Moerman, D. & Baillie, D. Genetics 91, 95-104 (1979).-   9. Benian, G., et al. Genetics 134, 1097-1104 (1993).-   10. Proud, C. TIBS 20, 241-246 (1995).-   11. Epstein H., et al. J. Mol. Biol. 90 291-300 (1974).-   12. Karn, J., et al. Proc. Natl. Acad. Sci. (U.S.A.) 80, 4253-4257    (1983).-   13. Doniach, T. & Hodgkin J. A. Dev. Biol. 106, 223-235 (1984).-   14. Spence, A., et al. Cell 60, 981-990 (1990).-   15. Krause, M., et al. Cell 63, 907-919 (1990).-   16. Chen, L., et al. Development, 120, 1631-1641 (1994).-   17. Dibb, N. J., et al. J. Mol. Biol. 205, 603-613 (1989).-   18. Sulston, J., et al. Dev. Biol. 100, 64-119 (1983).-   19. Sulston, J. & Horvitz, H. Dev. Biol. 82, 41-55 (1977).-   20. Draper B. W., et al. Cell 87, 205-216 (1996).-   21. Sulston, J., et al. Nature 356, 37-41 (1992).-   22. Matzke, M. & Matzke, A. Plant Physiol. 107, 679-685 (1995).-   23. Ratcliff, F., et al. Science 276, 1558-1560 (1997).-   24. Latham, K. Trends in Genetics 12, 134-138 (1996).-   25. Mello, C. & Fire, A. Methods in Cell Biology 48, 451-482 (1995).-   26. Clark, D., et al. Mol. Gen. Genet. 247, 367-378 (1995).-   27. Chalfie, M., et al. Science 263, 802-805 (1994).-   28. Studier, F., et al. Methods in Enzymology 185, 60-89 (1990).-   29. Bowerman, B., et al. Cell 68, 1061-1075 (1992).-   30. Mello, C. C., et al. Cell 70, 163-176 (1992).-   31. van der Keyl, H., et al. Develop. Dynamics 201, 86-94 (1994).-   32. Goeddel, D. V. Gene Expression Technology, Academic Press, 1990.-   33. Kriegler, M. Gene Transfer and Expression, Stockton Press, 1990.-   34. Murray, E. J. Gene Transfer and Expression Protocols, Humana    Press, 1991.

TABLE 1 Effects of sense, antisense, and mixed RNAs on progeny ofinjected animals. Gene and Segment Size Injected RNA F1 Phenotype unc-22unc-22 null mutants: strong twitchers^(7, 8) unc22A^(a) exon 21-22 742sense wild type antisense wild type sense + antisense strong twitchers(100%) unc22B exon 27 1033 sense wild type antisense wild type sense +antisense strong twitchers (100%) unc22C exon 21-22^(b) 785 sense +antisense strong twitchers (100%) fem-1 fem-1 null mutants: female (nosperm)¹³ fem1A exon 10^(c) 531 sense hermaphrodite (98%) antisensehermaphrodite (>98%) sense + antisense female (72%) fem1B intron 8 556sense + antisense hermaphrodite (>98%) unc-54 unc-54 null mutants:paralyzed^(7, 11) unc54A exon 6 576 sense wild type (100%) antisensewild type (100%) sense + antisense paralyzed (100%)^(d) unc54B exon 6651 sense wild type (100%) antisense wild type (100%) sense + antisenseparalyzed (100%)^(d) unc54C exon 1-5 1015 sense + antisense arrestedembryos and larvae (100%) unc54D promoter 567 sense + antisense wildtype (100%) unc54E intron 1 369 sense + antisense wild type (100%)unc54F intron 3 386 sense + antisense wild type (100%) hlh-1 hlh-1 nullmutants: lumpy-dumpy larvae¹⁶ hlh1A exons 1-6 1033 sense wild type (<2%lpy-dpy) antisense wild type (<2% lpy-dpy) sense + antisense lpy-dpylarvae (>90%)^(e) hlh1B exons 1-2 438 sense + antisense lpy-dpy larvae(>80%)^(e) hlh1C exons 4-6 299 sense + antisense lpy-dpy larvae(>80%)^(e) hlh1D intron 1 697 sense + antisense wild type (<2% lpy-dpy)myo-3 driven GFP transgenes^(f) myo-3::NLS::gfp::lacZ makes nuclear GFPin body muscle gfpG exons 2-5 730 sense nuclear GFP-LacZ pattern ofparent strain antisense nuclear GFP-LacZ pattern of parent strainsense + antisense nuclear GFP-LacZ absent in 98% of cells lacZL exon12-14 830 sense + antisense nuclear GFP-LacZ absent in >95% of cellsmyo-3::MtLS::gfp makes mitochondrial GFP in body muscle gfpG exons 2-5730 sense mitochondrial GFP pattern of parent strain antisensemitochondrial GFP pattern of parent strain sense + antisensemitochondrial GFP absent in 98% of cells lacZL exon 12-14 830 sense +antisense mitochondrial GFP pattern of parent strain

Legend of Table 1

Each RNA was injected into 6-10 adult hermaphrodites (0.5-1×10⁶molecules into each gonad arm). After 4-6 hours (to clear pre-fertilizedeggs from the uterus) injected animals were transferred and eggscollected for 20-22 hours. Progeny phenotypes were scored upon hatchingand subsequently at 12-24 hour intervals.

a: To obtain a semi-quantitative assessment of the relationship betweenRNA dose and phenotypic response, we injected each unc22A RNApreparation at a series of different concentrations. At the highest dosetested (3.6×10⁶ molecules per gonad), the individual sense and antisenseunc22A preparations produced some visible twitching (1% and 11% ofprogeny respectively). Comparable doses of ds-unc22A RNA producedvisible twitching in all progeny, while a 120-fold lower dose ofds-unc22A RNA produced visible twitching in 30% of progeny.

b: unc22C also carries the intervening intron (43 nt).

c: fem1A also carries a portion (131 nt) of intron 10.

d: Animals in the first affected broods (laid at 4-24 hours afterinjection) showed movement defects indistinguishable from those of nullmutants in unc-54. A variable fraction of these animals (25-75%) failedto lay eggs (another phenotype of unc-54 null mutants), while theremainder of the paralyzed animals were egg-laying positive. This mayindicate partial inhibition of unc-54 activity in vulval muscles.Animals from later broods frequently exhibit a distinct partialloss-of-function phenotype, with contractility in a subset of body wallmuscles.

e: Phenotypes of hlh-1 inhibitory RNA include arrested embryos andpartially elongated L1 larvae (the hlh-1 null phenotype) seen invirtually all progeny from injection of ds-hlh1A and about half of theaffected animals from ds-hlh1B and ds-hlh1C) and a set of less severedefects (seen with the remainder of the animals from ds-hlh1B andds-hlh1C). The less severe phenotypes are characteristic of partial lossof function for hlh-1.

f: The host for these injections, PD4251, expresses both mitochondrialGFP and nuclear GFP-LacZ. This allows simultaneous assay for inhibitionof gfp (loss of all fluorescence) and lacZ (loss of nuclearfluorescence). The table describes scoring of animals as L1 larvae.ds-gfpG caused a loss of GFP in all but 0-3 of the 85 body muscles inthese larvae. As these animals mature to adults, GFP activity was seenin 0-5 additional bodywall muscles and in the eight vulval muscles.

TABLE 2 Effect of injection point on genetic inhibition in injectedanimals and their progeny. dsRNA Site of injection Injected animalphenotype Progeny Phenotype None gonad or body cavity no twitching notwitching None gonad or body cavity strong nuclear & mitochondrial GFPstrong nuclear & mitochondrial GFP unc22B Gonad weak twitchers strongtwitchers unc22B Body Cavity Head weak twitchers strong twitchers unc22BBody Cavity Tail weak twitchers strong twitchers gfpG Gonad lowernuclear & mitochondrial GFP rare or absent nuclear & mitochondrial GFPgfpG Body Cavity Tail lower nuclear & mitochondrial GFP rare or absentnuclear & mitochondrial GFP lacZL Gonad lower nuclear GFP rare or absentnuclear GFP lacZL Body Cavity Tail lower nuclear GFP rare or absentnuclear GFP

TABLE 3 C. elegans can respond in a gene-specific manner toenvironmental dsRNA. Germline GFP-Transgene Bacterial Food MovementPhenotype Expression BL21(DE3) 0% twitch <1% female <1% faint GFPBL21(DE3) 0% twitch 43% female <1% faint GFP [fem-1 dsRNA] BL21(DE3) 85%twitch  <1% female <1% faint GFP [unc22 dsRNA] BL21(DE3) 0% twitch <1%female 12% faint GFP [gfp dsRNA]

TABLE 4 Effects of bathing C. elegans in a solution containing dsRNA.dsRNA Biological Effect unc-22 Twitching (similar to partial loss ofunc-22 function) pos-1 Embryonic arrest (similar to loss of pos-1function) sqt-3 Shortened body (Dpy) (similar to partial loss of sqt-3function)

In Table 2, gonad injections were carried out into the GFP reporterstrain PD4251, which expresses both mitochondrial GFP and nuclearGFP-LacZ. This allowed simultaneous assay of inhibition with gfp(fainter overall fluorescence), lacZ (loss of nuclear fluorescence), andunc-22 (twitching). Body cavity injections were carried out into thetail region, to minimize accidental injection of the gonad; equivalentresults have been observed with injections into the anterior region ofthe body cavity. An equivalent set of injections was also performed intoa single gonad arm. For all sites of injection, the entire progeny broodshowed phenotypes identical to those described in Table 1. This includedprogeny produced from both injected and uninjected gonad arms. Injectedanimals were scored three days after recovery and showed somewhat lessdramatic phenotypes than their progeny. This could in part be due to thepersistence of products already present in the injected adult. Afterds-unc22B injection, a fraction of the injected animals twitch weaklyunder standard growth conditions (10 out of 21 animals). Levamisoletreatment led to twitching of 100% (21/21) of these animals. Similareffects were seen with ds-unc22A. Injections of ds-gfpG or ds-lacZLproduced a dramatic decrease (but not elimination) of the correspondingGFP reporters. In some cases, isolated cells or parts of animalsretained strong GFP activity. These were most frequently seen in theanterior region and around the vulva. Injections of ds-gpG and ds-lacZLproduced no twitching, while injections of ds-unc22A produced no changein GFP fluorescence pattern.

While the present invention has been described in connection with whatis presently considered to be practical and preferred embodiments, it isunderstood that the invention is not to be limited or restricted to thedisclosed embodiments but, on the contrary, is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

Thus it is to be understood that variations in the described inventionwill be obvious to those skilled in the art without departing from thenovel aspects of the present invention and such variations are intendedto come within the scope of the present invention.

1-39. (canceled) 40: A method to inhibit expression of a target gene ina cell comprising synthesis of a first ribonucleic acid (RNA) and secondRNA in the cell, wherein the first and second RNAs hybridize to eachother to form a double-stranded structure in the cell, the first RNAconsisting essentially of a ribonucleotide sequence which corresponds toa nucleotide sequence of the target gene and the second RNA consistingessentially of a ribonucleotide sequence which is complementary to thenucleotide sequence of the target gene, wherein said first and saidsecond RNA are synthesized in the cell in an amount sufficient toinhibit the expression of the target gene, and wherein the target geneis an endogenous gene. 41: A method to inhibit expression of a targetgene in a cell comprising introducing into said cell double-strandedribonucleotide (RNA) comprising a first RNA consisting essentially of aribonucleotide sequence which corresponds to a nucleotide sequence ofthe target gene and a second RNA which consists essentially of aribonucleotide sequence which is complementary to the nucleotidesequence of the target gene and wherein said first and second RNAs arepresent in an amount sufficient to inhibit the expression of the targetgene. 42: The method of claim 40 or claim 41 wherein the target gene isan endogenous gene or a transgene. 43: The method of claim 42 whereinthe expression of the target gene is inhibited in the cell in vitro. 44:The method of claim 40 or claim 41 wherein the target gene expression isreduced by at least 10%. 45: The method of claim 40 and claim 41 whereinthe cell is an animal cell. 46: The method of claim 40 or claim 41wherein the cell is a plant cell. 47: A kit comprising reagents forinhibiting expression of a target gene in a cell, wherein said kitcomprises a means for introduction of a ribonucleic acid (RNA) into thecell in an amount sufficient to inhibit expression of the target gene,and wherein the RNA has a double-stranded structure with and identicalnucleotide sequence as compared to a portion of the target gene. 48: Amethod of inhibiting expression of a target gene in a cell in a mammalcomprising providing at least one ribonucleic acid (RNA) to the cell inan amount sufficient to inhibit the expression of a target gene, whereinsaid RNA is provided to the cell by synthesizing said RNA in said cell,wherein the RNA comprises or forms a double-stranded structurecontaining a first strand consisting essentially of a ribonucleotidesequence which corresponds to a nucleotide sequence of the target geneand a second ribonucleotide sequence which is complementary to thetarget gene, wherein the first and the second ribonucleotide sequencesare complementary sequences that hybridize to each other to comprise orform said double-stranded structure, and wherein the RNA comprising orforming the double-stranded structure inhibits expression of targetgene. 49: The method of claim 48, wherein said RNA is transcribed froman expression construct. 50: The method of claim 48, wherein saiddouble-stranded structure is formed by a single self-complementary RNAstrand comprising the first and second ribonucleotide sequences. 51: Themethod of claim 50, wherein said single self-complementary RNA strand istranscribed from an expression construct. 52: A method of inhibiting theexpression of a target gene in a mammalian cell, comprising contactingsaid mammalian cell with an expression construct, wherein saidexpression construct comprises an inverted duplication for a segment ofthe target gene, wherein said segment of the target gene comprises anucleotide sequence substantially identical to at least one portion ofthe target gene, wherein a promoter drives expression of saidinverted-duplication, and wherein said inverted-duplication forms adouble-stranded RNA structure which inhibits expression of the targetgene. 53: The method of claim 52, wherein the double-stranded RNAstructure is partially double-stranded. 54: An expression constructcomprising an inverted-duplication for a segment of a target gene in amammalian cell, wherein said segment of a target gene comprises anucleotide sequence substantially identical to at least a portion of thetarget gene, wherein a promoter drives expression of saidinverted-duplication, and wherein said inverted-duplication forms adouble-stranded RNA structure which is capable of inhibiting expressionof the target gene in a mammalian cell. 55: An expression constructcomprising a nucleotide sequence comprising a regulatory region, whereinsaid nucleotide sequence is capable of transcribing a singleself-complementary RNA strand which forms a double-stranded RNAstructure, and wherein said double-stranded RNA structure is capable ofinhibiting expression of a target gene in a mammalian cell.